Engagement of multicomponent immunoreceptors such as the T cell antigen receptor (TCR) results in rapid activation of multiple protein tyrosine kinases (PTKs) including Lck, Fyn, ZAP-70 and Itk. These PTKs then phosphorylate a number of enzymes and adapter molecules involved in a complex signaling cascade. Our studies have focused on a critical substrate of the PTKs, LAT (linker for activation of T cells), a 36-38kD integral membrane protein. LAT is a critical transmembrane adaptor protein. We have performed studies to characterize how LAT is phosphorylated and binds a number of critical signaling molecules, thus bringing these other adaptor molecules and enzymes to the plasma membrane in the vicinity of the activated TCR. Biochemical, biophysical, genetic and microscopic techniques are currently employed to study the characteristics of LAT-based signaling complexes. A critical pathway activated after TCR engagement and primarily dependent on the LAT molecule is the ERK pathway. A number of our older studies demonstrated how interaction of the enzyme phospholipase C gamma with a particular phosphorylated tyrosine of LAT results in activation of an enzyme cascade leading to ERK activation. Nonetheless in some T cells of mice bearing a mutant form of LAT incapable of binding this phospholipase C isoform, ERK activation can be activated. Studies completed this year demonstrate that an additional adapter molecule, Bam32, is found in T cells. It also is coupled to pathways leading to ERK activation. The role of this molecule in T cell activation in normal and our mutant mice is under investigation. In addition to biochemical and genetic studies of signaling molecules the laboratory has developed new methods of visualizing T cell activation using confocal microscopy. Many of the signaling molecules involved in the early TCR-coupled activation process have been tagged with fluorescent markers and expressed in T cells. The group has used these methods to observe the process of the assembly of signaling molecules into signaling clusters at the site of T cell activation. In studies completed this year we demonstrated that following TCR activation and signaling cluster formation, signaling molecules are internalized in a process dependent on the Cbl molecule, an E3 ubiquitin ligase. An additional study used our imaging techniques to demonstrate how the recruitment of signaling molecules at the point of activation is dependent on small G proteins of the Rab family. The use of imaging techniques to probe intracellular signaling in live, single cells continues to reveal important new facts about T cell activation.